Insulators possess two basic properties; l) they disrupt enhancer-promoter interactions only when inserted between these control elements and 2) they prevent chromosomal position effects. As such, insulators impart limitations to regulatory elements within chromosomes. Two general models have been proposed for insulator function; those in which insulators organize changes in higher-order chromatin structure to block regulatory interactions (domain boundary model) and those in which insulator proteins engage in local interactions with transcriptional proteins to intercept control signals (decoy model). To elucidate the molecular mechanisms involved in insulator action, we will study the gypsy insulator, which was isolated from the gypsy retrotransposon. This insulator provides an excellent model, as it is the only known insulator whose function requires a single DNA binding protein, the Suppressor of Hairy-wing [Su(Hw)] protein, to bring insulator complexes to the chromosome and whose effects can be manipulated genetically. In addition to binding the gypsy insulator, the Su(Hw) protein associates with nearly 200 non-gypsy sites within euchromatin. We will distinguish between the two prevailing models of insulator function by defining the mechanisms involved in blocking enhancer-promoter communication and prevention of heterochromatic repression. These studies will determine the effects of the gypsy insulator on the expression of defined transgenes into which the insulator is placed. This approach was chosen over those that study the bulk properties of the Su(Hw) protein because the nature of the non-gypsy target sites is unknown. To understand the function of the Su(Hw) protein at these sites, the non-gypsy target sites will be isolated and we will determine whether they possess insulator function. Our data will elucidate mechanisms used to define regulatory interactions within the genome, thereby providing insights into fundamental questions of how enhancer-promoter interact and the effects of chromatin structure on gene expression. Finally, our studies will enhance the development of better strategies to deliver dependable gene expression in gene therapies, as insulators overcome a major obstacle confronting such technologies, chromosomal position effects.